Whole Exome Sequencing Revealed a Novel GJB1 Pathogenic Variant and a Rare BSCL2 Mutation in Two Iranian Large Pedigrees with Multiple Affected Cases of Charcot-Marie-Tooth

Charcot-Marie-Tooth disease (CMT) is the most common hereditary neuropathy of the peripheral nervous system with a wide range of severity and age of onset. CMT patients share similar phenotypes which make it often impossible to identify the disease types based on clinical presentation and electrophysiological studies alone. In recent years, novel genetic diagnostic approaches such as whole exome sequencing (WES) has provided a ground for accurate diagnosis of CMT through identification of the disease-causing mutation(s). In the present study, that approach was effectively employed. Two unrelated large pedigrees with multiple affected cases of various pattern of inheritance (one autosomal dominant and one X-linked) were included. Clinical and electrophysiological data were obtained. DNA sample from each pedigree’s proband was subjected to WES. Data analysis was performed using an in-house developed pipeline, adopted from GATK and ANNOVAR. Candidate variant segregation was evaluated by PCR-based Sanger sequencing. A known but extremely rare (unreported in the Middle Easterners) mutation in BSCL2 (c.C269T:p.S90L) as well as a novel hemizygous variant in GJB1 (c.G224C:p.R75P) were identified and segregations were confirmed by Sanger sequencing. This study supports effectiveness of WES for genetic diagnosis of CMT in undiagnosed families.

harcot-Marie-Tooth disease (CMT), characterized by the slow progressive weakness, impaired exteroception, distal muscular atrophy as well as foot deformity is a heterogeneous neuromuscular disease with a prevalence of nearly 1 in 2,500 in western countries (1).   (9,10). In this study, WES was employed to find the genetic causes of CMT in two distinct pedigrees, manifesting an AD or X-linked type of CMT, which had remained genetically undiagnosed with either MLPA or Sanger sequencing.

Clinical evaluation
The study was conducted in accordance with Kit (Agilent Technologies, CA, USA). Data analysis was performed using an in-house developed pipeline, adopted from GATK and ANNOVAR (11,12).

PCR-based Sanger sequencing
Candidate variant segregation from exome data was evaluated by PCR-based Sanger sequencing on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The specific primers were designed using Geneious software. Primers are as follows: ACTA1_fwd 5-

Family 1
The family pedigree is displayed in Figure 1a.  Table 1.
The proband (III-2) is a 19-year-old boy suffering from unsteady gait and frequent falling started at the age of 7; yet, he was able to walk independently. Neurological examination at the age of 19 revealed a steppage gait with mild spastic feature, thenar atrophy, scoliosis, foot deformities, equinovarus as well as muscular atrophy and weakness in the lower and upper limbs, which showed predominantly distal involvement.
Electrophysiological features of the proband were consistent with an axonal neuropathy (Table 1). To

Family 2
A five-generation pedigree, depicted in Figure   2a, is presented as the second family with five members, 3 males and 2 females, suffering from an apparently X-linked dominant CMT. No male-tomale transmission was observed in the pedigree and the phenotype was more severe in males compared to females.     Table 1.
With regards to the other family members, a less severe phenotype of foot and hand deformities was reported in proband's mother (III-6). The identified c.C269T:p.S90L mutation in our study is a particularly rare mutation and of low frequency in CMT2 (18). To our knowledge, this mutation has only been reported in Europeans (14,(19)(20)(21)(22), and Taiwanese (18) The CMT2 phenotype itself is strongly heterogeneous and presents a variable penetrance.
Conventional understanding is that genetic diagnosis of CMT2 could not be attained in 75% of the clinically diagnosed cases of CMT2 using common diagnostic methods (26). For a robust genetic diagnosis of CMT2 as well as CMT2 differentiation from Silver syndrome and dHMNV, seipin/BSCL2 mutation screening seems to be important, particularly in patients with apparently upper motor neuron and lower motor neuron involvement. Subsequently, clinicians would be able to make correlation between clinical phenotypes and molecular results.
In the second studied family, an X-linked dominant 1 CMT (CMTX1) was confirmed.  (27). In this study, we identified the novel c.G224C:p.R75P variant in GJB1. Arginine in the position 75 of Cx32 is highly conserved amongst all the members of the connexin family (28). The truncated connexin 32 does not assemble in a proper manner and entraps in the Golgi. Although the Golgi-retained truncated protein could be degraded by lysosomal proteolysis or be transferred from the Golgi to the ER to be degraded by proteasomes (29,30). The absence of functional protein at plasma membrane of peripheral nerves results in neuropathy.
Accurate mutational diagnosis of CMT is extremely important for genetic counseling and even prenatal diagnosis (2). Remarkably, plenty of rare genes/potential mutations are not being tested for everyone due to the costliness of conventional genetic testing which initially starts with genetic analysis of PMP22, GJB1, MPZ, and MFN2, worldwide (31). As a result, almost 50 percent of the patients remain genetically undiagnosed (26).
Moreover, recently exome-first approach has been used for precise diagnosis of phenotypic and genetic heterogeneous diseases (32). Here we declare the diagnostic utility and affordability of WES not only for the patients whom initial genetic screening by conventional methods has been negative, but also as a first approach for genetic testing of such a diverse and complex trait.